Although it is widely accepted that resting, latently-infected CD4+ T-cells are a cART-resistant reservoir for HIV, little attention has been devoted to alternative reservoir sites. Using an ultrasensitive Fluorescent in situ Hybridization (FISH)-based system for detecting HIV mRNA by flow cytometry we mapped the distribution of HIV in the lung of chronically infected, asymptomatic HIV-seropositive individuals and found the virus to be present at greater abundance in alveolar macrophages (AMs) than in lymphocytes. AMs are unusual in that they are long-lived cells derived from a self-sustaining cell lineage retained within the lung. Recently, we extended our study to a cohort of patients on cART and found that those AMs positive for HIV mRNA persisted at sustained levels despite some individuals having been on cART for up to 9 years. The goal of this proposal is to assess the significance of these HIV-positive AMs as a potential reservoir with the capacity to reconstitute peripheral viremia upon cessation or failure of cART, and to develop strategies for its eradication. 1. Molecular Characterization of HIV-infected AMs. We have modified our FISH detection platform to sort cells positive for HIV mRNA, enabling us to perform RNASeq to profile both host and viral transcriptomes, and DeepSeq analysis of viral genome insertion sites to map viral population structure. We propose defining the viral genomes from individuals on suppressive cART to compare with peripheral virus isolated from those individuals who subsequently go on to fail therapy to establish their relatedness. 2. Are HIV mRNA-positive AMs productively infected? We will determine whether HIV mRNA-positive AMs can generate infectious virions that could seed CD4+ T-cells and reconstitute viremia. We are re-engineering viral reporter cell lines for use in Malawi to detect production of infectious virions from infected AMs. 3. Are AMs in non-human primates (NHP) similarly infected with SIV? Our collaborators, Dr. Charles Scanga, University of Pittsburgh and Dr. Shelby O'Connor, University of Wisconsin, are conducting a NHP study of SIV/M.tb co-infection. We will analyze these macaques during early SIV infection to determine whether NHP infected with SIV exhibit efficient early seeding of the AM population. We will also perform FISH and RNASeq on these cells to compare with the human HIV transcriptomes. 4. Can HIV-positive AMs be driven to cell death in vitro? We will pursue novel strategies towards elimination of these cells. The critical difference between CD4+ T-cells and macrophages is that macrophages are not driven to cell death by the infection. We will manipulate in vitro-infected human monocyte-derived macrophages (HMDMs) and AMs by chemical perturbation to generate preliminary data for a future HTS for small molecules capable of driving HIV-infected AMs into programmed cell death.